Benzimidazole compounds

ABSTRACT

Benzimidazole-4-carboxamide compounds (I) which can act as potent inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase or PARP enzyme (EC 2.4.2.30), and which thereby can provide useful therapeutic compounds for use in conjunction with DNA-damaging cytotoxic drugs or radiotherapy to potentiate the effects of the latter. In formula (I), R and R&#39; may each be selected independently from hydrogen, alkyl, hydroxyalkyl (e.g. CH 2  CH 2  OH), acyl (e.g. acetyl or benzoyl) or an optionally substituted aryl (e.g. phenyl) or aralkyl (e.g. benzyl or carboxybenzyl) group. R is generally a substituted phenyl group in the most preferred compounds. The compounds may also be used in the form of pharmaceutically acceptable salts or pro-drugs. ##STR1##

The present invention relates to certain benzimidazole compounds thatare of interest as being at least potentially useful chemotherapeuticagents by virtue of an ability to inhibit the activity of the enzymepoly ADP-ribosyltransferase (EC 2.4.2.30), also known aspoly(ADP-ribose) polymerase, commonly referred to as ADPRT or PARP. Ingeneral, the latter abbreviation, PARP, will be used throughout thepresent specification.

BACKGROUND

At least in higher organisms, the enzyme poly ADP-ribosyltransferase isknown to catalyse a transfer of the ADP-ribose moiety from the oxidizedform, NAD⁺, of nicotinamide adenine dinucleotide to nuclear acceptorproteins so as to form homo ADP-ribose polymers, and this process hasbeen implicated in a number of cellular events such as, for example,repair of DNA damage, development of cellular differentiation,transformation of cells by oncogenes, and gene expression A commonfeature in a number of these processes is the formation and repair ofDNA strand breaks and the stage which involves the PARP enzyme appearsto be that of DNA ligase II-mediated strand rejoining. In the majorityof cases a role for poly ADP-ribosylation has been implicated by the useof inhibitors of the PARP enzyme, and this has led to suggestions thatsuch inhibitors, by interfering with the intracellular DNA repairmechanism, may have a useful chemotherapeutic role insofar as theyshould be able to modify treatment resistance characteristics andpotentiate or enhance the effectiveness of cytotoxic drugs inchemotherapy or of radiation in radiotherapy where a primary effect ofthe treatment is that of causing DNA damage in target cells, as forexample in many forms of antitumour therapy.

In this connection, several classes of PARP inhibitors are alreadyknown, including benzamide and various nicotinamide and benzamideanalogues, especially 3-substituted benzamides with small substituentgroups such as 3-amino, 3-hydroxy and 3-methoxy. PARP inhibitoryactivity of certain N-substituted benzamides has also been reported inEP-A-0305008 wherein it has also been proposed to use these compounds inmedicine for increasing the cytotoxicity of radiation or ofchemotherapeutic drugs.

Regarding this use of benzamide compounds as chemotherapeutic agents,various studies on such compounds that are known to exhibit PARPinhibitory activity have confirmed that they can potentiate thecytoxicity of a range of antitumour agents in vitro, for example,bleomycin and methylating drugs. More limited data has further indicatedthat such benzamide compounds can also potentiate the activity ofcytotoxic drugs in vivo, although the dose requirements have appeared tobe rather high (e.g. in the region of 0.5 g kg⁻¹ per dose for3-aminobenzamide) and there may be associated problems in preparingsatisfactory pharmaceutical formulations and in avoiding toxicitylimitations. Furthermore, a number of the known benzamide compounds havealso been shown clearly to have potential as radiosensitizers,increasing for example ionising radiation-induced tumour cell kill bothin vitro and in vivo, and it is believed that in many cases this effectis related to these compounds acting as PARP inhibitors and interferingwith DNA repair.

However, notwithstanding the existing data from in vitro and in vivostudies suggesting that PARP inhibitors have considerable potential asuseful chemotherapeutic agents which merit further clinical evaluation,for instance in connection with cancer therapy, currently availableknown PARP inhibitors are not considered as yet to be entirely suitableto represent candidate drugs and there remains a need to find anddevelop a greater range of compounds having potentially useful PARPinhibitory properties.

DISCLOSURE OF THE INVENTION

The present invention identifies a new range or ranges of compounds ofinterest as PARP inhibitors that can be useful in medicine, especiallywhen administered in conjunction with at least certain cytotoxic drugsor with radiotherapy for increasing the cytotoxic effectiveness thereof.In general, the compounds to which this invention relates comprisecertain benzimidazole derivatives, more particularlybenzimidazole-4-carboxamide compounds, as hereinbelow defined. By virtueof their structure it would appear that many such compounds areparticularly well adapted to compete with the natural substrate NAD+ forthe PARP enzyme.

More specifically, from one aspect, the invention resides in the use ofa compound as herein defined for the manufacture of a medical orveterinary preparation for use in therapy for inhibiting activity of theenzyme poly(ADP-ribose)polymerase or PARP (also known as ADP-ribosyltransferase or ADPRT), such enzyme inhibition constituting an element ofa therapeutic treatment, wherein said compound provides the active PARPenzyme inhibiting agent and comprises a benzimidazole-4-carboxamidehaving the general structural formula I ##STR2## or a pharmaceuticallyacceptable salt and/or pro-drug form thereof, characterised in that instructural formula I

R is selected from hydrogen, alkyl, hydroxyalkyl (e.g. CH₂ CH₂ OH), acyl(e.g. acetyl or benzoyl) and an optionally substituted aryl (e.g.phenyl) or aralkyl (e.g. benzyl or carboxybenzyl) group, and

R' is selected from hydrogen, alkyl, hydroxyalkyl (e.g. CH₂ CH₂ OH),acyl (e.g. acetyl or benzoyl) and an optionally substituted aryl (e.g.phenyl) or aralkyl (e.g. benzyl or carboxybenzyl) group.

The invention also provides for use in therapy, as active pharmaceuticalsubstances, especially but not exclusively as PARP inhibitors,benzimidazole compounds having the general structural formula I ##STR3##(or a pharmaceutically acceptable salt and/or pro-drug form thereof),with substituents as defined above except for provisos that R does notrepresent 4'-methanesulphonyloxy-2'-methoxyphenyl or4'-methanesulphonylamino-2'-methoxyphenyl and does not represent aphenyl group having a substituent which is an alkylsulphenyl,alkylsulphinyl, alkanesulphonyl or alkylsulphoximino group, analkylsulphoximino group substituted at the nitrogen atom by an alkanoyl,alkylsulphonyl or hydroxycarbonyl-alkylenecarbonyl group, an ethoxy orn-propoxy group each of which is substituted in the terminal position byan alkylsulphenyl, alkylsulphinyl, alkanesulphonyl or alkylsulphoximinogroup, an alkoxycarbonylamino or an N-alkylaminocarbonylamino group andR' is not an optionally substituted aralkyl group and does not include abiphenyl or substituted biphenyl group.

The invention further provides novel benzimidazole compounds having thegeneral structural formula I (or a pharmaceutically acceptable saltand/or pro-drug form thereof), with substituents as defined immediatelyabove except for the further proviso that R does not represent anunsubstituted aryl group such as phenyl.

Alkyl groups when present as such or as a moiety in other groups willgenerally be composed of 1-8 carbon atoms, preferably 1-6 carbon atoms,and more usually 1-4 carbon atoms. In particular, when R and/or R' is analkyl group this will generally be C₁₋₆ alkyl, such as for examplemethyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl or cyclohexyl. WhenR and/or R' is or includes a phenyl group this may be substituted,especially in the 4 (para) position but alternatively or additionally inthe 2-position and/or 3-position for instance, by various substituentsincluding hydroxy, alkoxy (methoxy or ethoxy for example), cyano,carboxy, amide, tetrazole, amino or substituted amino, CW₃ (e.g. CF₃) orW where W is halogen.

In cases where R' is hydrogen or alkyl preferred compounds of structuralformula I include compounds in which R is phenyl or benzyl having atleast one substituent in the benzene ring which is selected fromhydroxy, alkoxy, NO₂,N₃, NR₅ R₆ (R₅ and R₆ each being independentlyhydrogen, alkyl or alkoxy), NHCOR₃ (R₃ being alkyl or aryl), CO₂ R₄ (R₄being H or alkyl), an amide (e.g. CONH₂), tetrazole, alkyl,hydroxyalkyl, CW₃ or W (W being halogen), and CN.

More particularly, where R represents a substituted phenyl group havingthe structural formula II ##STR4## R₁, R₂ and R₉ may be each selectedindependently from H, hydroxy, alkoxy, NO₂, N₃, NR₅ R₆ (R₅ and R₆ eachbeing independently hydrogen, alkyl or alkoxy), NHCOR₃ (R₃ being alkylor aryl), CO₂ R₄ (R₄ being H or alkyl), an amide (e.g. CONH₂),tetrazole, alkyl, hydroxyalkyl, CW³ or W (W being halogen), and CN.

The invention also includes a process for preparing a compound ofstructural formula I as specified above wherein R represents anoptionally substituted phenyl group having the structural formula II,said process comprising the steps of reacting an alkyl2,3-diaminobenzoate with an aryl acid chloride, treating the productwith acetic acid at an elevated temperature to bring about benzimidazolering formation, and reacting with liquid ammonia to form the amidederivative.

Where R' represents a substituted phenyl group having the structuralformula III ##STR5## R₇, R₈ and R₁₀ may be each selected independentlyfrom H, hydroxy, alkoxy, NO₂, N₃, NR₅ R₆ (R₅ and R₆ each beingindependently hydrogen, alkyl or alkoxy), NHCOR₃ (R₃ being alkyl oraryl), CO₂ R₄ (R₄ being H or alkyl), an amide (e.g. CONH₂), tetrazole,alkyl, hydroxyalkyl, CW₃ or w (W being halogen), and CN.

Compounds of structural formula I as hereinabove defined which have anaromatic ring that includes a CN substituent may often also beparticularly useful as intermediates in making other compounds inaccordance with the invention since a cyano substituent can generally beconverted, using standard methodology, into a variety of otherfunctional groups, including amine, carboxyl, amide and tetrazole.

Within the ranges of benzimidazole compounds disclosed herein, preferredmembers which are of particular interest include

(a) 2-methylbenzimidazole-4-carboxamide;

(b) benzimidazole-4-carboxamide;

(c) 2-phenylbenzimidazole-4-carboxamide;

(d) 2-(4'-methoxyphenyl)benzimidazole-4-carboxamide;

(e) 2-(4'-trifluoromethylphenyl)benzimidazole-4-carboxamide;

(f) 2-(4'-hydroxyphenyl)benzimidazole-4-carboxamide;

(g) 2-trifluoromethylbenzimidazole-4-carboxamide;

(h) 2-(4'-methoxyphenyl)-N-methylbenzimidazole-4-carboxamide;

(i) 2-(4'-nitrophenyl)benzimidazole-4-carboxamide;

(j) 2-(4'-cyanophenyl)benzimidazole-4-carboxamide;

(k) 2-(3'-trifluoromethylphenyl)benzimidazole-4-carboxamide;

(l) 2-(3'-methoxyphenyl)benzimidazole-4-carboxamide;

(m) 2-(4'-methoxyphenyl)-1-N-benzoylbenzimidazole-4-carboxamide,

(n) 2-(4'-aminophenyl)benzimidazole-4-carboxamide

(o) 2-(2'-trifluoromethylphenyl)benzimidazole-4-carboxamide,

(p) N-carboxybenzyl-2-(4'-methoxyphenyl)-benzimidazole-4-carboxamide.

In the above-mentioned compounds of this invention wherein there is anelectron-rich aromatic ring, it is believed that in at least some casesthe carboxamide group may be constrained in a fixed conformation,particularly favourable for presenting the compound as an inhibitor ofNAD⁺ binding to the PARP enzyme, by an intramolecular hydrogen bondbetween an imidazole ring nitrogen atom and one of the hydrogen atoms ofthe carboxamide group.

As already indicated, the invention also embraces or extends to methodsof preparing compounds as hereinbefore defined (including intermediatesin some cases) and to the therapeutic use of such compounds in treatingmammals. This includes their use for making medical or veterinarypreparations or pharmaceutical formulations containing an effective PARPinhibitory amount of the active compound for administration to a patientin conjunction with a cytotoxic drug or radiotherapy in order toincrease the cytotoxic effectiveness of the latter. Such preparations orformulations may be made up in accordance with any of the methods wellknown in the art of pharmacy for administration in any suitable manner,for example orally, parenterally (including subcutaneously,intramuscularly or intravenously), or topically, the mode ofadministration, type of preparations or formulation and the dosage beinggenerally determined by the details of the associated cytotoxic drugchemotherapy or radiotherapy that is to be enhanced.

In making up such pharmaceutical formulations in the form of sterileliquid preparations for parental use for instance, a predeterminedtherapeutically effective non-toxic amount of the particular compoundconcerned may be dissolved in phosphate buffered saline and thepreparations may be presented in unit dosage form and contained insealed ampoules ready for use. In general, at least in aqueous solution,concentrations not greater than 200 mg/ml will be preferred, but theamount and dosage routine required for optimum effectiveness will ofcourse vary and is ultimately at the discretion of the medical orveterinary practitioner treating the mammal concerned in each particularcase. Where the compound is to be used in conjunction with a cytotoxicdrug, the latter in some cases may be administered simultaneously andmay be conveniently incorporated in the same pharmaceutical formulationor composition.

As indicated, the compounds according to this invention have at leastpotential as PARP inhibitors, and in vitro tests hereinafter describedhave demonstrated positive pharmacological activity which it is believedreflects the activity to be found in vivo in the course of therapeuticclinical use.

It will be understood that where reference is made in this specificationto compounds of formula I such reference should be construed asextending also to their pharmaceutically acceptable salts and to otherpharmaceutically acceptable bioprecursors (pro-drug forms) whererelevant. The term "pro-drug" is used in the present specification todenote modified forms or derivatives of a pharmacologically activecompound which biodegrade in vivo and become converted into said activecompound after administration, especially oral or intravenousadministration, in the course of therapeutic treatment of a mammal. Suchpro-drugs are commonly chosen because of an enhanced solubility inaqueous media which helps to overcome formulation problems, and also insome cases to give a relatively slow or controlled release of the activeagent.

A satisfactory pro-drug must generally be a water-soluble derivativewhich is non-toxic and reasonably stable in solution at physiological pHbut which will biodegrade or convert, e.g. by enzymatic degradation orby an enviromental pH change, to the active compound at the locationrequired following administration in the course of therapy. For thebenzimidazole compounds of the present invention, pro-drug forms mayconveniently be provided by carbamate or amino acid derivatives, e.g.glycine or other amino-acid carbamate derivatives, or by phosphatederivatives. Phosphate derivatives may be susceptible to enzymicdephosphorylation in viva and are presently preferred, especiallywater-soluble ammonium or alkali metal phosphate salts. These may oftenbe conveniently prepared from compounds of structural formula I havingat least one hydroxyl group substituent, e.g. in an aromatic ringcomponent of R, by reacting with a dibenzyl phosphonate, preferably inthe presence of a tertiary base such as N,N-diisopropylethylamine.

In cases where R is phenyl (or benzyl) and where it is necessary to havea substituent other than hydroxyl, e.g. NO₂, CO₂ H, CN etc. at the 4'position in order to give satisfactory PARP inhibitory activity, ahydroxyl substituent amenable to phosphorylation or other pro-drugmodification may be provided at another aromatic ring position, e.g. atthe 3' position.

In all the water-soluble pro-drug forms presently envisaged thephosphate, carbamate or other water-solubilizing pro-drug moiety will bea component of R or R' in structural formula I.

It should also be understood that where any of the compounds referred tocan exist in more than one enantiomeric form, all such forms, mixturesthereof, and their preparation and uses are within the scope of theinvention.

BRIEF DESCRIPTION OF THE DRAWING

The invention is illustrated by reference to the accompanying drawingwherein:

FIG. 1 is a graph showing results obtained using the invention.

DESCRIPTION OF EXAMPLES OF PREFERRED EMBODIMENTS

The following examples and descriptions of stages in synthetic routes ofpreparation of various preferred compounds of interest serve to furtherillustrate the present invention, but should not be construed in any wayas a limitation thereof.

In the first example (EXAMPLE 1), the preparation is described ofvarious intermediate compounds required for the preparation ofbenzimidazole compounds in accordance with the present invention whichare described in EXAMPLES 2 to 6.

Example 1 Preparation of Intermediate Compounds

(a) 3-Nitrophthalamic Acid

3-Nitrophthalic anhydride (10.0 g, 50 mmol) was added in portions over20 minutes to concentrated aqueous ammonia solution (15 ml), and themixture was stirred at 300° C. for a further 30 minutes. The crystallinemass of ammonium phthalamate, deposited upon cooling the pale yellowsolution, was collected and redissolved in a minimum amount of warmwater. Concentrated hydrochloric acid (4.5 ml) was added dropwise, withstirring, and the resulting paste was washed with water, and dried invacuo to give 3-nitrophthalamic acid as a fine white powder. (9.01 g,83%), m.p. 217° C. Found: C, 45.76; H, 2.79; N, 13.21. C₈ H₆ N₂ O₅requires C, 45.71; H, 2.86; N, 13.33%; v_(max) /cm⁻¹ 3466.52, 3321.84,1668.64, 1604.98, and 1525.89; δ_(H) (d₆ -DMSO, 200 MHz) 7.75 (1H, br s,CONH), 7.8 (1H, t, Ar-5H), 8.16 (1H, brs, CONH), 8.2 (1H, d, Ar-6H), 8.3(1H, d, Ar-4H); δ_(C) (d₆ -DMSO) 127.32, 130.06, 132.28, 133.49, 134.78,147.71, 166.25, and 166.60; m/z (EI) 192 (M⁺ -1), 177, 149, 103, 75.

(b) 2-Amino-3-nitrobenzoic Acid (3-nitroanthranilic acid)

To a stirred solution of potassium hydroxide (24.1 g) in water (110 ml)at 0° C. was added bromine (2.46 ml), followed by 3-nitrophthalamic acid10 g, 47.62 mmol). The reaction mixture was stirred for 3 hours at 60°C., cooled to room temperature, and stirred for a further 12 hours. Theorange precipitate was collected, redissolved in a minimum amount ofwater, and acidified by the dropwise addition of concentratedhydrochloric acid. Recrystallisation of the resulting yellow solid fromhot water afforded 3-nitroanthranilic acid as yellow microcrystals (6.42g, 74%), m.p. 208-209° C. Found: C, 45.83; H, 3.07; N, 15.21. C₇ H₆ N₂O₄ requires C, 46.15; H, 3.29; N, 15.38%; v_(max) /cm⁻¹ 3476.17,3344.99, 3094.21 and 1687.93; δ_(H) (d₆ -DMSO, 200MHz) 6.76-6.84 (1H, t,Ar-5H), 8.29-8.41 (2H, dd, Ar-4/6H), 8.60 (2H, s, Ar--NH₂), 13.4-14.0(1H, br s, Ar--CO₂ H); δ_(C) (d₆ -DMSO) 113.19, 131.97, 140.02(Ar-4/5/6CH), 115.02 (Ar--C--NH₂), 132.77 (Ar-C-CO₂ H), 147.09(Ar--C--NO₂), 168.95 (Ar--CO₂ H); m/z (EI) 182 (M⁺), 164.

(c) Methyl 2-amino-3-nitrobenzoate

Hydrogen chloride gas was bubbled through a solution of2-amino-3-nitrobenzoic acid (0.5 g, 2.75 mmol) in methanol (40 ml) for15 minutes at 0° C. The reaction mixture was heated under reflux for 5hours, and allowed to cool to room temperature over a further 12 hours,whereupon methyl 2-amino-3-nitrobenzoate was deposited as a yellow solid(417 mg, 77%), m.p. 95-96° C. Found: C, 49.09: H, 3.78; N, 14.03. C₈ H₈N₂ O₄ requires C, 48.98; H, 4.08; N, 14.29%; v_(max) /cm⁻¹ 3452.5,3316.9, 1702, and 1253.7; δ_(H)) d₆ -DMSO, 200 MHz) 3.95 (3H, s, OCHH₃),6.79-6.87 (1H, t, Ar-5H), 8.28-8.33 (1H, dd, Ar-4H), 8.41-8.46 (1H, dd,Ar-6H), 8.45-8.46 (2H, br s, Ar--NH₂); m/z (EI) 196 (M⁺), 164, 118, 90,63.

(d) 2,3-Diaminobenzoic Acid

Palladium on carbon catalyst (10% Pd, ˜200 mg) was added cautiously, asa slurry in methanol (10ml), to a solution of 3-nitroanthranilic acid(2.44 g, 13 mmol) in methanol (120 ml), and the mixture was stirredunder a hydrogen atmosphere for 2 hours until the absorption of gasceased. The catalyst was removed by filtration through Celite, and thefiltrate was evaporated to dryness under reduced pressure to afford thecrude product. Purification by column chromatography on silica gel, withdichloromethane:methanol (4:1) as eluent, gave 2,3-diaminobenzoic acidas a red solid (1.34 g, 66%). v_(max) /cm⁻¹ 3433.73, 2882.02, 2602.30and 1658.99; δ_(H) (d₆ -DMSO, 200 MHz) 5.8-7.4 (4H, br s, 2×NH₂), 6.45(1H, t, Ar-5H), 6.75 (1H, d, Ar-4H), 7.20 (1H, d, Ar-6H); δ_(C) (d₆-DMSO) 110.31, 115.45, 118.33, 120.55, 135.03, 140.36, 170.68; m/z (EI)152 (M⁺), 134, 106, 79.

(e) Methyl 2,3-diaminobenzoate

A solution of 2,3-diaminobenzoic acid (0.2 g, 1.32 mmol) in methanol (40ml) was saturated with hydrogen chloride as described above, and themixture was subsequently heated under reflux for 2 hours. The solidresidue obtained on evaporation of the solvent was dissolved in water,and the solution was adjusted to pH 7.0 with sodium hydrogen carbonate.After extraction with ethyl acetate (2×30 ml), the combined organiclayers were dried (MgSO₄), and the solvent was removed to give methyl2,3-diaminobenzoate as a brown oil which solidified on trituration withpetrol (40/60) (121.6 mg, 56%), m.p. 62-63° C. Found: C, 58.35; H, 5.80;N, 16.69. C₈ H₁₀ N₂ O₂ requires C, 57.83; H, 6.02; N, 16.87%; δ_(H) (d₆-DMSO), 200 MHz) 3.87 (3H, s, OCH₃). 4.90 (2H, br s, Ar-2-NH₂), 6.32(2H, br s, Ar-3-NH₂), 6.46-6.54 (1H, t, Ar-5H), 6.80-6.84 (1H, dd,Ar-4H), 7.18-7.23 (1H, dd, Ar-6H); m/z (EI) 166 (M⁺), 134, 106, 79.

Methyl 2,3-diaminobenzoate was also prepared by reduction of methyl2-amino-3-nitrobenzoate as follows: a solution of methyl2-amino-3-nitrobenzoate (284 mg, 1.45 mmol) in methanol (40 ml),containing palladium on carbon catalyst (10% Pd, ˜50 mg), was stirredunder hydrogen for 24 hours. The solution was filtered through Celite toremove the catalyst, and the solvent was evaporated in vacuo to affordthe methyl ester as a brown solid. (180 mg, 75%) identical to methyl2,3-diaminobenzoate prepared above.

(f) Methyl 2-amino-3-N-benzoylaminobenzoate

A solution of benzoyl chloride (38.4 μl, 0.331 mmol) in tetrahydrofuran(5 ml) was added dropwise to a solution of methyl 2,3-diaminobenzoate(50 mg, 0.301 mmol) in dry tetrahydrofuran (5 ml), containingtriethylamine (46 μl) and 4-dimethylaminopyridine (1.8 mg, 5 mol %).After stirring the mixture for 24 hours at 45° C., solvents wereevaporated, and the crude product was purified by column chromatographyon silica gel, with petrol (40/60); ethyl acetate (3:2) as eluent.Recrystallisation from ethyl acetate-petrol (40/60), gave the titlecompound as white crystals. (60 mg, 74%); δ_(H) (d₆ -DMSO, 200 MHz) 3.95(3H, s, OCH₃), 6.64 (2H, br s, Ar--NH₂), 6.69-6.77 (1H, t, Ar-5H),7.46-7.50 (1H, d, Ar-4H), 7.59-7.70 (3H, m, Ph-3 and Ph-3' 4H),7.81-7.85 (1H, d, Ar-6H), 8.11-8,14 (2H, d, Ph-2H and Ph-2'H), 9.8-9.9(1H, br, s, Ar--NHCO); m/z (EI) 270 (M⁺), 253, 105.

(g) Methyl 2-amino-3-N-(4'-methoxybenzoyl)aminobenzoate

To a solution of methyl 2,3-diaminobenzoate (460 mg, 2.77 mmol) in drytetrahydrofuran (20 ml) was added 4-methoxybenzoyl chloride (378 μl,2.77 mmol), triethylamine (385.5 μl, 2.77 mmol), and4-dimethylaminopyridine (17 mg, 5 mol %). The reaction mixture wasstirred at room temperature overnight, yielding an insoluble precipitatethat was collected by filtration. The filtrate was evaporated underreduced pressure and the residual solid was redissolved in boilingmethanol, and hot filtered to remove the insoluble material. The solventwas removed in vacuo, and the solid residue was combined with thepreviously collected precipitate. Recrystallisation from aqueousmethanol afforded white crystals of the title compound. (513.2 mg, 62%);mp 179-180° C.; Found: C, 64.26; H, 5,31; N, 9.17. C₁₆ H₁₆ N₂ O₄requires C, 64.0; H, 5.33; N, 9.33; v_(max) /cm⁻¹ 3425.54, 3341.54,3277.84, 1699.24, 1632.12, 1251.11; δ_(H) (d₆ DMSO, 200 MHz) 3.92 (3H,s, OMe), 3.94 (3H, s, OMe), 6.59 (2H, s, Ar--NH₂), 6.68-6.75 (1H, t,Ar-5H), 7.13-7.17 (2H, d, J=8.8, Ph-3/3'H), 7.43-7.46 (1H, d, Ar-4H),7.79-7.83 (1H, d, Ar-6H), 8.07-8.12 (2H, d, J=8.8, Ph-3.3'H), 9.7 (1H,br s, --NHCO--); δ_(C) (d₆ DMSO) 51.98, 55.76, 110.62, 113.79, 114.67,125.0, 126.84, 129.12, 130.14, 133.20, 147.36, 162.21, 165.74, 168.33;m/z (EI) 300 (M⁺), 135, 107, 77.

(h) Methyl 2-phenylbenzimidazole-4-carboxylate

A solution of methyl 2-amino-3-N-benzoylaminobenzoate (6.3 mg, 0.023mmol) in glacial acetic acid (0.5 ml) was stirred under reflux for 15minutes. After cooling, the solvent was removed under reduced pressureto afford the title compound; δ_(H) (d₆ -DMSO, 200 MHz) 4.09 (3H, s,OCH₃), 7.40-7.48 (1H, t, Ar-30 5H), 7.64-7.70 (3H, m, 2-Ph-3H and3'-Ph-4H), 7.93-7.97 (1H, d, Ar-4H), 8.06-8.10 (1H, d, Ar-6H), 8.39-8.41(2H, d, 2-Ph-2/2'H), 12.4-12.5 (1H, br, s, Ar--NHCO).

Example 2 Benzimidazole-4-carboxamide (Compound NU1066)

(a) 1st Stage--Preparation of Benzimidazole-4-carboxylic Acid (CompoundNU1067)

A mixture of 2,3-diaminobenzoic acid (0.5 g, 3.29 mmol) and formic acid(405 μl, 9.87 mmol) in hydrochloric acid (4M, 10 ml) was heated underreflux for one hour. The precipitate which formed on cooling wascollected, redissolved in boiling methanol, and decolorised withactivated charcoal. Evaporation of the solvent gavebenzoxazole-4-carboxylic acid as a white powder (407.9 mg, 77%)

Found: C, 46.11; H, 3.63; N, 13.27. C₈ H₆ N₂ O₂.HCl.0.5 H₂ O requires C,46.28; H, 3.88; N, 13.49%; δ_(H) (d₆ -DMSO, 200 MHz) 7.7-7.8 (1H, t,Ar-5H), 8.2-8.3 (2H, dd, Ar-4/6H), 9.65 (1H, s, imidazole-2H).

(b) 2nd Stage--Preparation of Benzimidazole-4-carboxamide (CompoundNU1066)

A suspension of benzimidazole-4-carboxylic acid (3.97.4 mg, 2.45 mmol)in thionyl chloride (10 ml) was heated under reflux for 3.5 hours, andthe thionyl chloride was removed by vacuum distillation. The residualsolid was suspended in dry tetrahydrofuran (10 ml) and added dropwise toconcentrated aqueous ammonia (50 ml) with stirring over 30 minutes.Excess solvent was removed in vacuo, and the residue was dissolved in aminimum volume of water and extracted with ethyl acetate (2×20 ml). Thesolid recovered on evaporation of the combined organic layers wasdissolved in hydrochloric acid (0.1M, 10 ml) and the insolubleprecipitate was removed by filtration. The aqueous filtrate wascarefully adjusted to pH 9 in increments of 1 pH unit, and ethyl acetateextractions (10 ml) were undertaken at each step. The combined extractswere dried (MgSO₄) and the solvent was evaporated. Recrystallisationfrom ethyl acetate furnished benzimidazole-4-carboxamide (50 mg, 13%)

Found: C, 59.95; H, 3.90; N, 24.59. C₉ H₇ N₃ O requires C, 59.63; H,4.35; N, 26.09%; uv/nm 210, 270, 291; v_(max) /cm⁻¹ 3321.84, 3150.16,1747.73, 1680.21; δ_(H) (d₆ -DMSO, 200 MHz) 7.4 (1H, t, Ar-5H), 7.8-8.0(3H, dd, Ar-4/6H), 8.5 (1H, br s, imidazole-2H), 9.4 (1H, br s, CONH),13.1 (1H, br s, CONH); m/z (EI) 161 (M⁺), 141, 116, 99.

Example 3 2-Methylbenzimidazole-4-carboxamide (Compound NU1064)

(a) 1st stage--Preparation of 2-Methylbenzimidazole-4-carboxylic Acid

Acetic acid (0.23 ml) was added to a solution of 2,3-diaminobenzoic acid(200 mg, 1.32 mmol) in hydrochloric acid (4M, 3.2 ml) and the mixturewas refluxed for 1 hour. Solvents were evaporated and the residual solidwas redissolved in boiling methanol (5 ml) and decolorised withactivated charcoal. Removal of the solvent furnished2-methylbenzimidazole-4-carboxylic acid as an amorphous white solid(167.5 mg, 72%); δ_(H) (d₆ -DMSO) 2.9 (3H, s, imidazole-2-CH₃), 7.6-7.8(1H, t, Ar-5H) 8.1 (2H, d, Ar-4/6H); m/z (EI 176 (M⁺), 158, 130.

(b) 2nd stage--Preparation of 2-Methylbenzimidazole-4-carboxamide(Compound NU1064)

A suspension of 2-methylbenzimidazole-4-carboxylic acid (500 mg, 2.84mmol) in thionyl chloride (10 ml) was heated under reflux for 2 hours,and the thionyl chloride was removed by vacuum distillation. The solidresidue was redissolved in dry tetrahydrofuran, and added dropwise toconcentrated aqueous ammonia solution (50 ml) over 30 minutes, withstirring. The solvent was removed under vacuum, and the solid residuewas redissolved in a minimum of hot water, filtered, and extracted withethyl acetate (2×30 ml). Evaporation of the solvent afforded a brownsolid which was recrystallised from ethyl acetate to give the titlecompound as a white solid (70.1 mg, 14%)

Found: C, 61.47; H, 4.96; N, 23.39. C₉ H₉ N₃ O requires C, 61.71; H,5.14; N, 24.0%; uv/nm 209, 270; v_(max) /cm⁻¹ 3296.77, 3071.07, 1913.63,1859.62, 1805.60; δ_(H) (d₆ -DMSO, 200 MHz) 2.68 (3H, s,imidazole-2-CH₃), 7.30-7.38 (1H, t, Ar-5H), 7.72-7.46 (1H, d, Ar-4H),7.86-7.90 (1H, d, Ar-6H), 7.72-7.90 (1H, br s, imidazole-NH), 9.4 (1H,br s, CONH), 12.8 (1H, brs, CONH); m/z (EI) 175 (M⁺), 158, 130.

Example 4 2-Phenylbenzimidazole-4-carboxamide (Compound NU1070)

(a) 1st Stage--Preparation of 2-phenylbenzimidazole-4-carboxylic Acid

A mixture of 2,3-diaminobenzoic acid (0.1 g, 0.66 mmol), benzoic acid(80.2 mg, 0.66 mmol) and polyphosphoric acid (˜5 ml) was heated at150-160° C. for 30 minutes, and, after cooling, crushed ice (˜10 g) wasadded. Insoluble materials were removed from the dark solution byfiltration, and the filtrate was extracted with ethyl acetate (2×20 ml)to remove unreacted benzoic acid. The aqueous solution was cautiouslyneutralised with sodium hydroxide (10 M), filtered, and the filtrate wasextracted with ethyl acetate (2×30 ml). The combined extracts were dried(MgSO₄) and the solvent was evaporated. Chromatography on silica gel,with dichloromethane:methanol (85:15) as eluent, gave the title compound(31.2 mg, 20%) ; δ_(H) (d₆ -DMSO, 200 MHz) 7.4 (1H, t, Ar-5H), 7.62 (3H,br s, 3-Ph-4H and 3'-Ph-4H), 7.91 (1H, d, Ar-6H), 7.97 (1H, d, Ar-4H),8.39 (2H, d, Ph-2H and Ph2'-H); m/z (EI) 238 (M⁺), 220, 192, 77.

(b) 2nd Stage--Preparation of 2-phenylbenzimidazole-4-carboxamide(NU1070)

2-Phenylbenzimidazole-4-carboxylic acid (50 mg, 0.21 mmol) was dissolvedin dry tetrahydrofuran (10 ml) and thionyl chloride (16.8 μl, 0.231mmol) and DMF (0.05 ml) were added. The mixture was stirred at roomtemperature for 12 hours, when a white precipitate developed, and thesuspension was added dropwise to stirred aqueous ammonia (10 ml) over 10minutes. The mixture was stirred for a further 30 minutes, diluted withwater (20 ml), and neutralised with hydrochloric acid (4M). The whitesolid which was precipitated upon cooling, was collected by filtrationto afford 2-phenylbenzimidazole-4-carboxamide (31 mg, 62%); v_(max)/cm⁻¹ 3320, 3180, 1660 and 1600; δ_(H) (d₆ -DMSO, 200 MHz) 7.45 (1H, t,Ar-5H), 7.72 (3H, d, 3-Ph-4H), 7.87 (1H, d, Ar-4H), 7.97 (1H, br s,CONH), 7.99 (2H, d, Ar-6H), 8.38 (2H, d, Ph-2-H and Ph-2-H), 9.5 (1H, brs, CONH); m/z (EI) 237 (M⁺), 220, 192, 165, 77.

Example 5 2-(4'-Methoxypheny)benzimidazole-4-carboxamide (NU1076)

(a) 1st Stage--Preparation of Methyl 2-(4'-methoxyphenyl)benzimidazole-4-carboxylate Acetate Salt Methyl2-amino-3-N-(4'-methoxybenzoyl)benzoate (480 mg, 1.6 mmol) was dissolvedin glacial acetic acid (15 ml), and heated at 120°-130° C. for 30minutes. The solvent was removed and the solid residue was recrystalisedfrom ethyl acetate-petrol (40/60) to yield the product as a whitecrystalline solid. (409 mg, 75%); mp 141-142° C.; Found: C, 63.68; H,4,79; N, 7.88; C₁₆ H₁₄ N₂ O₃.CH₃ CO₂ H requires C, 63.16; H, 5.26; N,8.19; v_(max) /cm⁻¹ 3375.33, 1718.46, 1696.80, 1282.81, 1257.81,1257.34; δ_(H) (d₆ DMSO), 200 MHz) 2.02 (3H, s, CH₃ CO₂ H), 3.97 (3H, s,OMe), 4.09 (3H, s, OMe), 7.21-7.25 (2H, d, J=8.6, Ph-3/3'H), 7.39-7.46(1H, t, Ar-5H), 7.90-7.93 (1H, d, Ar-4H), 8.00-8.04 (1H, d, Ar-6H),8.36-8.40 (2H, d, J=8.6, Ph-2/2'H), 12.1 (1H, s, Imz-H), 12.3-12.4 (1H,br, s, CH₃ CO₂ H) ; δ_(C) (d₆ DMSO) 21.35, 52.37, 55.64, 114.41, 121.68,122.35, 124.34, 129.56, 153.63, 161.27, 166.13, 172.37; m/z (EI) 282 (M⁺--CH₃ CO₂ H), 250, 222, 77, 60, 43, 32.

(b) 2nd Stage--Preparation of2-(4'-Methoxypheny)benzimidazole-4-carboxamide (NU1076)

The acetate salt of methyl(2-(4'-methoxyphenyl)benzimidazole-4-carboxylate was dissolved in excessliquid ammonia and heated at 100° C. in a sealed pressure vessel at 40atmospheres overnight. The ammonia was allowed to evaporate, and thesolid residue was collected and washed with ice cold water (3×5 ml).Recrystallisation from aqueous methanol afforded the title compound(226.4 mg, 80%); mp 261-263° C.; Found: C, 66.07; H, 4.23; N, 15.29. C₁₅H₁₃ N₃ O₂. 0.2CH₃ OH requires C, 66.70; H, 5.08; N, 15.35; v_(max) /cm⁻¹3321.47, 3140.72, 1656.23, 1608.25, 1421.43, 1242.55; δ_(H) (d₆ DMSO,200 MHz) 3.96 (3H, s, OMe), 7.23-7.27 (2H, d, J=8.6, Ph-3/3'H),7.37-7.45 (1H, t, Ar-5H), 7.78-7.82 (1H, d, Ar-4H), 7.87 (1H, br s,Imz-H), 7.93-7.96(1H, d, Ar-6H), 8.27-8.31 (2H, d, J=8.6, Ph-2/2'H),9.4-9.5 (1H, br s, --CONH), 13.3-13.4 (1H, br s, --CONH); m/z (EI) 267(M⁺), 249, 222, 206, 77, 32.

Example 6 2-(4'-trifluoromethyl)benzimidazole-4-carboxamide (NU1077)

(a) 1st Stage--Preparation of Methyl2-amino-3-N-(4'-trifluoromethylbenzoyl)aminobenzoate

To a solution of methyl 2,3-diaminobenzoate (300 mg, 1.807 mmol) wasadded 4-trifluoromethylbenzoyl chloride (268.4 μl, 1.807 mmol),triethylamine (251.4 μl, 1.807 mmol) and 4-dimethylaminopyridine (11 mg,5 mol %), and the mixture was stirred at room temperature overnight. Thereaction solvent was removed under reduced pressure and the resultingsolid was washed with ethyl acetate. Recrystallisation twice frommethanol-water gave the title compound as a white solid. (83.6 mg, 14%);mp 180-181° C.; Found: C, 56.75; H, 3.50; N, 8.28. C₁₆ H₁₃ F₃ N₂ O₃requires C, 56.80; H, 3.85; N, 8.28; uv/nm 222; δ_(H) (d₆ DMSO, 200 MHz)3.93 (3H, s, OMe), 6.70-6.76 (3H, m, Ar-5H, Ar--NH₂), 7.46-7.49 (1H, d,Ar-4H), 7.81-7.85 (1H, d, Ar-6H), 7.99-8.03 (2H, d), 8.29-8.33 (2H, d),10.05 (1H, s, --NHCO--); m/z (EI) 338 (M⁺), 321, 289, 145, 32.

(b) 2nd Stage--Preparation of Methyl2-(4'-trifluoromethylphenyl)benzimidazole-4-carboxylate Acetate Salt

Methyl 2-amino-3-N-(4'-trifluoromethylbenzoyl) aminobenzoate (75.7 mg,0.224 mmol) was dissolved in glacial acetic acid (5 ml) and stirred at125° C. for 0.5 hour. The solvent was evaporated and the remaining whitesolid was washed with petrol (40/60) to yield the title compound. (59.6mg, 70%); mp 138-140° C.; Found: C, 56.78; H, 3.98; N, 7.36; C₁₆ H₁₁ F₃N₂ O₂ CH₃ CO₂ H requires C, 56.84; H, 3.94; N, 7.37. uv/nm 206, 319;δ_(H) (d₆ -DMSO, 200 MHz) 2.01 (3H, s, CH₃ CO₂ H), 7.44-7.52 (1H, t,Ar-5H), 7.97-8.14 (4H, m), 8.65-8.66 (2H, d), 12.1 (br s, Imidazole-NH),12.7-12.8 (1H, br s, CH₃ CO₂ H); m/z (EI) 320 )M⁺ --CH₃ CO₂ H), 301,288, 260, 145, 60, 43.

(c) 3rd Stage--Preparation of 2-(4'-trifluoromethyl)benzimidazole-4-carboxamide (NU1077)

The acetate salt of methyl2-(4'-trifluoro-methylphenyl)benzimidazole-4-carboxylate was dissolvedin excess liquid ammonia and heated at 100° C., in a sealed pressurevessel at 40 atmospheres, for 12 hours. The ammonia was allowed toevaporate, and the solid residue was washed with ice cold water (3×5ml). Recrystallisation from methanol-water yielded the product as finewhite needles. (19.1 mg, 48%); mp 301-305° C.; Found: C, 56.45; H, 3.50;N, 12.41. C₁₅ H₁₀ F₃ N₃ O.CH₃ OH requires C, 56.97; H, 4.18; N, 12.46;δ_(H) (d₆ -DMSO, 200 MHz) 7.45 (1H, t, Ar-5H), 7.88-7.92 (1H, d, Ar-4H),7.99 (1H, br s imidazole-NH), 8.03 (1H, d, Ar-6H); 8.06-8.10 (2H, d,J=8.1), 8.55-8.59 (2H, d, J=8.1), 9.3-9.4 (1H, br s, --CONH), 13.7-13.8(1H, br s, --CONH); m/z (EI) 288 (M⁺ --NH₃), 260, 69.

Example 7 2-(4'-Hydroxyphenyl)-1-H-benzimidazole-4-carboxamide (CompoundNU1085)

Under an argon atmosphere 1M boron tribromide in dichloromethane (3.8ml, 3.79 mmol) was transferred to a flask containing2-(4'-methoxyphenyl)benzimidazole-4-carboxamide (NU 1076 from Example 5)(202.4 mg, 0.758 mmol). The resulting solution was refluxed for 24 hoursusing an air condenser. The solvent was removed by distillation tocomplete dryness. The solid residue was treated with 10% NaOH (10 ml),followed by dropwise addition of concentrated hydrochloric acid toneutralise (pH 7). The white precipitate was collected by filtration anddissolved in ethyl acetate (10 ml). The organic solvent was washed withwater (2×3 ml), dried over MgSO₄, and the product was obtained byremoval of the solvent under reduced pressure. (109.5 mg, 57%). mp266-267° C.; Found C, 63.27, H 4.37; N 15.67; C₁₄ H₁₁ N₃ O₂. 0.75 MeOHrequires C, 63.04; H, 4.69; N, 15.76; v_(max) (cm⁻¹) 3424.01, 3384.16,3309.20, 3249.55, 3155.62, 1642.35, 1618.02, 1594.50, 1577.74; δ_(H)7.03-7.07 (2H, d, J=8.5), 7.34-7.42 (1H, t), 7.75-7.79 (1H, d), 7.85(1H, br s), 7.90-7.94 (1H, d), 8.15-8.19 (2H, d, J=8.5), 9.4-9.6 (1H, brs), 10.0-10.4 (1H, br s), 13.0-13.4 (1H, br s); m/z (EI) 253 (M⁺), 236,208, 93.

Example 8 2-(4'-Methoxyphenyl)-1-methylbenzimidazole-4-carboxamide(Compound NU1090)

2-(4'-Methoxyphenyl)benzimidazole-4-carboxamide (NU1076 from Example 5)(105.3 mg, 0.394 mmol) and powdered potassium hydroxide (22 mg, 0.394mmol) were suspended in acetone (4 ml) and stirred until all the solidshad dissolved. Methyl iodide (24.6 μl1, 0.394 mnol) was added and thereaction stirred at room temperature overnight. The solvent was removedunder reduced pressure and the white solid residue purified by columnchromatography with dichloromethane/methanol 95:5 to give fine whitecrystals of the title compound. (33.2 mg, 30%)

mp 289-292° C.; Found C, 68.62; H, 5.36; N, 14.67 C₁₆ H₁₅ N₃ O₂ RequiresC, 68.33; H, 5.34; N, 14.95; v_(max) (cm⁻¹) 3309.23, 3141.44, 1671.29,1605.30, 1255.08, δ_(H) 3.95 (3H, S), 4.02 (3H, s), 7.22-7.27 (2H, d),7.44-7.52 (1H, t), 7.86-8.00 (5H, m), 9.4 (1H, br s, NH); m/z (EI) 281(M⁺), 264, 250.

Example 9 2-(4'-Methoxyphenyl)-1-benzoylbenzimidazole-4-carboxamide(Compound NU1101)

A solution of 2-(4'-Methoxyphenyl)benzimidazole-4-carboxamide (NU1076from Example 5) (75.1 mg, 0.281 mmol) and powdered potassium hydroxide(15.8 mg, 0.281 mmol) was prepared in acetone (3 ml) and stirred untilall the solids had dissolved. Benzoyl chloride (32.6 μl, 0.281 mmol) wasadded and the solution stirred overnight at room temperature, with theproduction of a white precipitate. The solvents were removed underreduced pressure, and the white residue was purified by columnchromatography using dichloromethane/methanol 95:5. The resulting solidwas recrystallised from petrol 40/60/ethyl acetate to give the pureproduct as brilliant white prisms. (15.6 mg, 15%).

mp 207-210° C.; Found C, 70.45; H, 4.60; N, 10.99; C₂₂ H₁₇ N₃ O₃.0.25CH₃ OH Requires C, 70.45; H, 4.47; N, 11.08; v_(max) (cm³¹ 1) 3445.99,3318.55, 2922.99, 1689.79, 1666.36; δ^(H) 3.86 (3H, s, OCH₃), 7.02-7.06(2H, d), 7.50-7.65 (4H, m), 7.72-7.82 (3H, m), 7.88-7.92 (2H, d), 8.08(1H, s, CONH), 8.10-8.14 (1H, d), 9.1-9.2 (1H, br s, CONH); m/z (EI) 371(M⁺), 105.

FURTHER EXAMPLES

The following further examples, and also some of the examples alreadydescribed, make use of certain common standard procedures. Thesecomprise:

(1) Reaction of Methyl 2,3-diaminobenzoate with Aryl Acid Chlorides(Standard Procedure A)

(2) Benzimidazole Ring Formation by Acid Catalysed Cyclisation (StandardProcedure B)

(3) Amide Formation by Reaction with Liquid Ammonia (Standard ProcedureC)

The experimental details of these standard procedures are describedbelow:

Standard Procedure A

An ice/salt bath cooled solution of methyl 2,3-diaminobenzoate (1equivalent), dry triethylamine (1-1.5 equivalents) anddimethylaminopyridine (DMAP--5 mol %) in half the required volume of drytetrahydrofuran (THF) was prepared. The required acid chloride (1equivalent) was dissolved in the remaining dry tetrahydrofuran (THF) andadded to the cooled solution with stirring over 30 minutes. The reactionwas allowed to warm slowly to room temperature and was stirredovernight. The solvent was filtered to remove a precipitate which wassuspended in ethyl acetate, washed twice with water followed bysaturated brine, and dried with MgSO₄. The organic layer was added tothe reaction filtrate, and the solvent removed under reduced pressure.The solid residue was redissolved in ethyl acetate, washed twice withwater followed by saturated brine, and dried with MgSO₄. Removal of thesolvents under reduced pressure left a solid residue which was purifiedby column chromatography and/or recrystallisation from suitablesolvents.

Standard Procedure B

The starting material was dissolved in glacial acetic acid and plungedinto a pre-heated oil bath at 120° C. The solution was heated for theappropriate time and then allowed to cool to room temperature. Theacetic acid was removed under reduced pressure and the solid residuepurified by column chromatography and/or recrystallisation from suitablesolvents.

Standard Procedure C

The starting material was dissolved in a excess of freshly condensedliquid ammonia. This was heated to 800° C. within a sealed vessel,generating a pressure of 40 atmospheres, for 24 hours. The ammonia wasevaporated, and the solid residue obtained purified by columnchromatography and/or recrystallisation from suitable solvents.

Example 10 2-(4'-Cyanophenyl)-1-H-benzimidazole-4-carboxamide (NU1092)

(a) 1st Stage--Preparation of Methyl2-amino-3-N-(4'cyanobenzoyl)aminobenzoate

Following standard procedure A, methyl 2,3-diaminobenzoate (300 mg, 1.81mmol), triethylamine (251 μl, 1.81 mmol) and DMAP (11 mg) were dissolvedin THF (7.5 ml) and cooled. To this was added 4-cyanobenzoyl chloride(299 mg, 1.81 mmol) dissolved in THF (7.5 ml). The product was purifiedby column chromatography, dichloromethane/methanol 99:1, followed byrecrystallisation from boiling methanol. (196 mg, 37%)

mp 198-202° C.; v_(max) (cm³¹ 1) 3486.40, 3374.02, 3245.61, 2231.25,1688.04, 1646.65; δ_(H) 3.93 (3H, s, CO₂ CH₃), 6.68-6.76 (1H, t), 6.72(2H, br s, NH₂), 7.45-7.49 (1H, d) 7.81-7.86 (1H, d), 8.11-8.15 (2H, d,J=8.4), 8.25-8.29 (2H, d, J=8.4), 10.01 (1H, br s, NH); m/z (EI) 295(M⁺), 278, 263, 246, 130, 102.

(b) 2nd Stage--Preparation of Methyl2-(4'-cyanopohenyl)-1-H-benzimidazole-4-carboxylate Following standardprocedure B, methyl 2-amino-3-N-(4'-cyanobenzoyl) aminobenzoate (301 mg,1.02 mmol) from 1st stage was heated in glacial acetic acid (10 ml). Theproduct was obtained by recrystallising twice using petrol 40/60/ethylacetate. (203 mg, 72%)

mp 195-198° C.; v_(max) (cm⁻¹) 3447.66, 2228.84, 1691.90, 1288.11 δ_(H)4.09 (3H, s, CO₂ CH₃), 7.44-7.53 (1H, t), 7.97-8.01 (1H, d), 8.10-8.13(2H, d, J=8.4), 8.58-8.62 (2H, d, J=8.4), 12.8 (1H, br s); m/z (EI) 277(M⁺), 245, 217

(c) 3rd Stage--Preparation of2-(4'-Cyanophenyl)-1-H-benzimidazole-4-carboxamide (NU1092) Followingstandard procedure C, methyl2-(4'-cyanophenyl)-1-H-benzimidazole-4-carboxylate (169.5 mg, 0.612mmol) was treated with ammonia under pressure. The crude product wasrecrystallised from boiling methanol to yield the title compound pure aswhite crystals. (116.5 mg, 73%)

mp>310° C.; Found C, 67.81; H, 3.89; N, 20.87; C₁₅ H₁₀ N₄ O.0.2MeOHRequires C, 67.95; H, 4.05; N, 20.85; v_(max) (cm⁻¹) 3332.27, 3274.86,3177.98, 2230.85, 1658.54, 1608.10; δ_(H) 7.45-7.49 (1H, t); 7.87-7.91(1H, d), 7.91 (1H, br s), 7.98-8.02 (1H, d); 8.13-8.17 (2H, d, J=8.3),8.50-8.54 (2H, d, J=8.3), 9.2-9.4 (1H, br s), 13.6-13.8 (1H, br s); m/z(EI) 262 (M⁺), 245, 217, 102.

Example 11 2-(4'-Nitrophenyl)-1-H-benzimidazole-4-carboxamide (NU1091)

(a) 1st Stage--Preparation of Methyl2-amino-3-N-(4'-nitrobenzoyl)aminobenzoate

Following standard procedure A, methyl 2,3-diaminobenzoate (300 mg,1.807 mmol), dry triethylamine (276.6 μl, 1.988 mmol) and DMAP (11 mg)were dissolved in dry THF (12 ml). To this was added 4-nitrobenzoylchloride (335.2 mg, 1.807 mmol) in dry THF (12 ml). Columnchromatography with dischloromethane/methanol 99:1 followed byrecrystallisation from methanol gave the product pure.

mp 196-197° C.; Found C, 57.08; H, 3.78; N, 13.25; C₁₅ H₁₃ N₃ O₅Requires C, 57.14; H, 4.12; N, 13.33; v_(max) (cm⁻¹) 3382.31, 3293.01,3256.56, 1702.05, 1657.83, 1525.37; δ_(H) 3.94 (3H, s, CO₂ CH₃),6.70-6.78 (1H, t), 6.66 (2H, br s, NH₂), 7.48-7.51 (1H, d), 7.83-7.87(1H, d), 8.33-8.38 (2H, d, J=8.8), 8.46-8.51 (2H, d, J=8.8), 10.15 (1H,br s, NH); m/z (EI) 315 (M⁺), 297, 265, 165.

(b) 2nd Stage--Preparation of Methyl2-(4'-Nitrophenyl)-1-H-benzimidazole-4-carboxylate

Following standard procedure B, methyl2-amino-3-N-(4'-nitrobenzoyl)aminobenzoate (340.2 mg, 1.08 mmol) washeated in glacial acetic acid (10 ml) for 15 minutes. The product wasobtained pure by recrystallisation from methanol. (208 mg, 65%).

mp 208-210° C.; Found C, 60.69; H, 3.57; N, 13.96; C₁₅ H₁₁ N₃ O₄Requires 60.61; H, 3.70; N, 14.14; v_(max) (cm⁻¹) 3433.70, 1720.14,1601.84, 1513.07; δ_(H) 4.21 (3H, s, CO₂ CH₃), 7.57-7.65 (1H, t),8.10-8.12 (1H, d), 8.23-8.27 (1H, d), 8.60-8.64 (2H, d, J=8.8),8.78-8.82 (2H, d, J=8.8), 13.04 (1H, br s, NH); m/z (EI) 297 (M⁺), 265.

(c) 3rd Stage--Preparation of2-(4'-Nitrophenyl)-1-H-benzimidazole-4-carboxamide (NU1091)

Following standard procedure C, methyl2-(4'-nitrophenyl)-1-H-benzimidazole-4-carboxylate was dissolved inliquid ammonia and heated under constant volume in a pressure vessel.The product was purified by column chromatography fromdichloromethane/methanol 99:1 and recrystallised from methanol.

mp>310° C.; δ_(H) 7.48-7.56 (1H, t), 7.90-7.94 (1H, d), 8.00 (1H, s,NH), 8.00-8.04 (1H, d), 8.52-8.56 (2H, d, J=8.8), 8.60-8.64 (2H, d,J=8.8), 9.3-9.4 (1H, br s, NH), 13.8-14.0 (1H, br s, NH)

Example 12 2-(3'-Trifluoromethylphenyl)-1-H-benzimidazole-4-carboxamide(NU1093)

(a) 1st Stage--Preparation of Methyl2-amino-3-N-(3'-trifluoromethylbenzoyl)aminobenzoate

Following standard procedure A, methyl 2,3-diaminobenzoate (200 mg,1.205 mmol), dry triethylamine (704 μl, 5.06 mmol) anddimethylaminopyridine (DMAP, 7.3 mg) were dissolved in dry THF (7.5 ml).To this was added 3-trifluoromethylbenzoyl chloride (183 μl, 1.205 mmol)in dry THF (7.5 ml). Column chromatography with dichloromethane/methanol99:1 removed impurities and the more polar product was eluted withdichloromethane/methanol 97:3. Recrystallisation from methanol gave theproduct as a white solid. (160.4 mg, 26%).

mp 157-159° C.; Found C, 57.14; H, 3.57; N, 8.10; C₁₆ H₁₃ F₃ N₂ O₃Requires C, 56.80; H, 3.85; N, 8.28; v_(max) (cm⁻¹) 3368.48, 3283.82,2953.87, 1705.98, 1650.77, 1250.02; δ_(H) 3.93 (3H, S, CO₂ CH₃),6.69-6.77 (1H, t), 6.73 (2H, s, NH₂), 7.45-7.49 (1H, d), 7.82-7.92 (2H,m), 8.06-8.10 (1H, d), 8.40-8.44 (1H, d), 8.48 (1H, s, 2'-H), 10.1 (1H,s, NH); m/z (EI) 338 (M⁺), 320, 288, 260, 173, 145.

(b) 2nd Stage--Preparation of Methyl2-(3'-trifluoromethylohenyl)-1-H-benzimidazole-4-carboxylate AcetateSalt

Following standard procedure B, a glacial acetic acid (6 ml) solution ofmethyl 2-amino-3-N-(3'-trifluoromethylbenzoyl)aminobenzoate was heatedfor 15 minutes. Removal of the solvent under reduced pressure followedby drying at high vacuum yielded the product as a pure white solid.(154.2 mg, 96%).

mp 105-107° C.; Found C, 56.93; H, 3.78; N, 7.32; C₁₆ H₁₁ F₃ N₂ O₂.CH₃CO₂ H Requires C, 56.84; H, 3.95; N, 7.37; v_(max) (cm⁻¹) 3438.30,3339.14, 2959.13, 1707.99, 1328.24, 1313.53; δ_(H) 2.01 (3H, s, CH₃ CO₂H), 4.09 (3H, s, CO₂ CH₃), 7.44-7.51 (1H, t), 7.79-8.13 (4H, m),8.71-8.75 (1H, d), 8.82 (1H, s), 11.8-12.2 (1H, br s), 12.8-13.0 (1H, brs); m/z (EI) 320 (M⁺ --CH₃ CO₂ H), 288, 260.

(c) 3rd Stage--Preparation of2-(3'-trifluoromethylphenyl)-1-H-benzimidazole-4-carboxamide (CompoundNU1093)

Following standard procedure C, the acetate salt of methyl2-(3'-trifluoromethylphenyl)-1-H-benzimidazole-4-carboxylate (134.8 mg,0.358 mmol) was treated with excess liquid ammonia in a sealed vessel.The product was purified by recrystallisation from methanol, to yieldoff-white needles. (78 mg, 72%).

mp 268-270° C.; Found C, 57.68; H, 3.82; N, 12.96; C₁₅ H₁₀ F₃ N₃O.0.6CH₃ OH Requires C, 57.74; H, 3.82; N, 12.95; v_(max) (cm⁻¹)3488.83, 3348.86, 3176.45, 1667.66, 1600.93, 1329.63; δ_(H) 7.44-7.52(1H, t), 7.88-8.04 (5H, m), 8.66-8.70 (1H, d), 8.70 (1H, s, 2'H), 9.3(1H, br s, NH), 13.6 (1H, br s, NH); m/z (EI) 305 (M⁺), 288, 260, 145.

Example 13 2-(3'-Methoxyphenyl)-1-H-benzimidazole-4-carboxamide (NU1098)

(a) 1st Stage--Preparation of Methyl2-amino-3-N-(3'-methoxybenzoyl)aminobenzoate

Following standard procedure A, a solution of methyl 2,3-diaminobenzoate(670.3 mg, 4.038 mmol), dry triethylamine (842.6 μl, 6.057 mmol) andDMAP (25 mg) in dry THF (20 ml) was prepared. A solution of3-methoxybenzoyl chloride (567 μl, 6.038 mmol) in dry THF (20 ml) wasadded to this. The resulting solid residue was purified by columnchromatography using dichloromethane/methanol 99:1 and the product wasobtained pure after two recrystallisations from petrol 40/60/ethylacetate. (282.6 mg, 23%)

mp 124-125° C.; Found C, 63.90; H, 5.11; N, 9.24; C₁₆ H₁₆ N₂ O₄ RequiresC, 64.0; H, 5.33; N, 9.33; v_(max) (cm⁻¹) 3386.19, 3292.38, 1697.97,1586.87, 1520.79, 1250.27; δ_(H) 3.92 (3H, s), 3.93 (3H, s), 6.61 (2H,s, NH₂), 6.68-6.76 (1H, t), 7.22-7.27 (1H, d), 7.44-7.47 (1H, d),7.49-7.57 (1H, t), 7.66 (1H, s, 2'-H), 7367-7.71 (1H, d), 7.79-7.84 (1H,d), 9.8 (1H, s, NH); m/z (EI) 300 (M⁺), 283, 135, 107.

(b) 2nd Stage--Preparation of Methyl2-(3'-methoxyphenyl)-1-H-benzimidazole-4-carboxylate Acetate Salt

Following standard procedure B, methyl2-amino-3-N-(3'-methoxybenzoyl)aminobenzoate (356.9 mg, 1.19 mmol) waswarmed in glacial acetic acid (12 ml. The removal of the solvent underreduced pressure followed by recrystallisation with petrol 40/60/ethylacetate afforded the title compound pure. (235.6 mg, 58%)

mp 93-94° C.; Found C, 62.66; H, 5.13; N, 8.06; C₁₆ H₁₄ N₂ O₃.CH₃ CO₂ HRequires C, 63.16; H, 5.26; N, 8.18; v_(max) (cm⁻¹) 3453.23, 3375.10,1706.75, 1257.40; δ_(H) 1.99 (3H, s, CH₃ CO₂ H), 3.96 (3H, s), 4.06 (3H,s), 7.15-7.21 (1H, d), 7.38-7.46 (1H, t), 7.51-7.59 (1H, t), 7.91-8.00(3H, m), 8.04-8.08 (1H, d), 12.0 (1H, s), 12.5 (1H, s); m/z (EI) 282 (M⁺--CH₃ CO₂ H), 250.

(c) 3rd Stage--Preparation of2-(3'-Methoxychenyl)-1-H-benzimidazole-4-carboxamide (NU1098)

Following standard procedure C, a liquid ammonia solution of methyl2-(3'-methoxyphenyl)-1-H-benzimidazole-4-carboxylate (203 mg, 0.596mmol) was heated under constant volume. The solid residue wasrecrystallised from methanol to give the pure product (73.5 mg, 46%).

mp 223-225° C.; Found C, 67.52; H, 4.91; N, 15.62; C₁₅ H₁₃ N₃ O₂Requires C, 67.42; H, 4.87; N, 15.73; v_(max) (cm⁻¹) 3408.59, 3388.94,3168.65, 1662.05, 1625.86, 1603.39; δ_(H) 3.99 (3H, s, OCH₃), 7.22-7.27(1H, d), 7.43-7.51 (1H, t), 7.58-7.66 (1H, t), 7.85-8.01 (5H, m),9.4-9.5 (1H, br s), 13.5 (1H, br s); m/z (EI) 267 (M⁺), 250.

Example 14 2-(2'-trifluoromethylphenyl)-1-H-benzimidazole-4-carboxamide(NU1104)

(a) 1st Stage--Preparation of Methyl2-amino-3-N-(2'-trifluoromethylbenzoyl)aminobenzoate

Following standard procedure A, methyl 2,3-diaminobenzoate (564 mg, 3.4mmol) in a THF (20 ml) solution with triethylamine (709 μl, 5.1 mmol)and dimethylaminopyridine (21 mg) was stirred and to this was added aTHF (20 ml) solution of 2-trifluoromethylbenzoyl chloride. The resultingoily residue was absorbed onto silica and then subjected to columnchromatography with dichloromethane/methanol 99:1 as eluant. The productwas obtained pure after recrystallisation from petrol 40/60/ethylacetate. (303 mg, 26%).

mp 163-166° C.; Found C, 56.91; H, 3.75; N, 8.29; C₁₆ H₁₃ F₃ N₂ O₃Requires C, 56.80; H, 3.85; N, 8.28; v_(max) (cm⁻¹) 3329.85, 3243.90,2955.52, 1696.66, 1663.58, 1312.69; δ_(H) 3.94 (3H, s, CO₂ CH₃), 6.58(2H, s, NH₂), 6.74-6.82 (1H, t), 7.57-7.62 (1H, d), 7.79-8.03 (5H, m),10.0 (1H, s, NH); m/z (EI) 338 (M⁺), 321, 289, 173, 145.

(b) 2nd and 3rd Stages--Preparation of2-(2'-trifluoromethyl)-1-H-benzimidazole-4-carboxamide (NU1104)

Upon subjecting the product of the 1st stages successively to standardprocedures B and C, the title compound was obtained.

Example 15 2-(4'-Aminochenyl)-1-H-benzimidazole-4-carboxamide (NU1103)

(a) 1st Stage--Preparation ofMethyl-2-amino-3-N-(4'-aminobenzoyl)aminobenzoate

Methyl-2-amino-3-N-(4'-nitrobenzoyl)aminobenzoate (from 1st stage ofExample 11) was suspended in methanol (40 ml) and a slurry of 10%palladium catalyst on activated carbon (˜50 mg) in methanol (10 ml) wasadded to this with stirring under argon. The solution wasatmospherically hydrogenated for 2 hours. After filtration throughCELITE (Regd. TM) to remove the catalyst the product was obtained byremoval of the solvent under reduced pressure to give a white solidwhich was dried under high vacuum. (204.1 mg, 92%).

mp 197-200° C.; Found C, 62.95; H, 5.30; N, 14.39; C₁₅ H₁₅ N₃ O₃Requires C, 63.16; H, 5.26; N, 14.73; v_(max) (cm⁻¹) 3472.55, 3374.96,3348.97, 3283.31, 1694.80, 1613.91; δ_(H) 3.94 (3H, s, CO₂ CH₃), 5.87(2H, s, NH₂), 6.54 (2H, s, NH₂), 6.68-3.73 (2H, d), 6.73-6.76 (1H, t),7.42-7.47 (1H, d), 7.78-7.82 (2H, d), 9.4 (1H, s, NH); m/z (EI) 285(M⁺), 267, 235, 207, 120, 92.

(b) 2nd Stage--Preparation of Methyl2-(4'-aminophenyl)-1-H-benzimidazole-4-carboxylate Acetate Salt

Following standard procedure B, the treatment of methyl2-amino-3-N-(4'-aminobenzoyl)aminobenzoate (186.5 mg, 0.654 mmol) withhot glacial acetic acid (8 ml) for 30 minutes yielded the title compoundfollowing recrystallisation from petrol 40/60/ethyl acetate. (113.4 mg,91%)

mp 162-164° C.; Found C, 62.60; H, 5.04; N, 12.73; C₁₅ H₁₃ N₃ O₂.CH₃ CO₂H Requires C, 62.39; H, 5.20; N, 12.84; v_(max) (cm⁻¹) 3450.66, 3369.25,3254.20, 1692.41, 1607.56, 1253.80; δ_(H) 2.02 (3H, s, CH₃ CO₂ H), 4.08(3H, S, CO₂ CH₃), 5.81 (2H, s, NH₂), 6.75-6.80 (2H, d, J=8.6), 7.32-7.40(1H, t), 7.83-7.86 (1H, d), 7.93-7.97 (1H, d), 8.08-8.13 (2H, d, J=8.6),11.9 (1H, s), 12.1 (1H, br s); m/z (EI) 267 (M⁺ --CH₃ CO₂ H), 235, 207,92, 60.

(c) 3rd Stage--Preparation of 2-(4⁴⁰-Aminophenyl)-1-H-benzimidazole-4-carboxamide (NU1103)

Following standard procedure C, the acetate salt of methyl2-(4'-aminophenyl)-1-H-benzimidazole-4-carboxylate (113 mg, 0.346 mmol)was treated with liquid ammonia under pressure for 24 hours. The puretitle compound was isolated with column chromatography of the crudematerial using dichloromethane/methanol 90:10 (21.4 mg, 25%)

mp 237-240° C.; δ_(H) 5.90 (2H, S, NH₂), 6.79-6.83 (2H, d, J=8.3),7.31-7.39 (1H, t), 7.71-7.75 (1H, d), 7.84 (1H, s, NH), 7.88-7.92 (1H,d), 8.00-8.04 (2H, d, J=8.3), 9.5-9.6 (1H, br s, NH), 13.0 (1H, br s,NH).

ASSAY FOR PARP INHIBITORY ACTIVITY

Compounds of the present invention, particularly those detailed in thepreceding Examples, have been tested in vitro for activity as PARPinhibitors using the following methods and materials.

In principle, the PARP assay used relies upon activating endogenous PARP(as hereinafter described) in cells containing exogenous [³² p]-NAD⁺introduced therein by suspending the cells in a solution of [³² p]-NAD⁺to which they have been rendered permeable in an initial pre-treatmentstep. The poly(ADP-ribose) which is then synthesised by the enzyme canbe precipitated by tri-chloracetic acid (TCA) and the amount ofradio-labelled ³² p incorporated therein measured, e.g. using ascintillation counter, to give a measure of the activity of the PARPunder the particular conditions of the experiment. By repeating theexperiment following the same procedure, and under the same conditions,in the presence of each compound to be tested the reduction in enzymeactivity, representative of the inhibitory effect of the test compound,can then be ascertained from the reduction, if any, of the amount of [³²p] measured in the TCA precipitated poly(ADP-ribose).

The results of this assay may be expressed in terms of percentageinhibition or reduction in activity for one or more differentconcentrations of each compound tested, or it may be expressed in termsof that concentration of the tested compound which reduces the enzymeactivity by 50%, i.e. the IC₅₀ value. Thus, with a range of differentcompounds a set of comparative values for inhibitory activity can beobtained.

In practice, L1210 murine leukaemia cells have been used as a source ofthe PARP enzyme after being rendered permeable to exogenous [³² P]NAD byexposure to hypotonic buffer and cold shock. In the preferred techniquewhich has been found to give exact and reproducible results, a definedamount of a small synthetic oligonucleotide, in particular a singlestrand oligonucleotide having the palindromic sequence CGGAATTCCG, isintroduced into the cell suspension for activating the PARP enzyme. Thisoligonucleotide sequence snaps back on itself to form a double-strandedmolecule with a single blunt end and provides an effective substrate foractivation of PARP. Its behaviour as a potent activator of the enzymewas confirmed in the tests carried out.

The experimental protocol adopted, in which a synthetic oligonucleotideas mentioned above is introduced as a specific activator of PARP,discriminates between PARP and other mono-ADP-ribosyltransferases in thecells. Thus, introduction of such synthetic oligonucleotides causes a 5to 6 fold stimulation in the radioactive label incorporated and this isattributable solely to PARP activity.

Further details of the assay are given below.

MATERIALS

The materials used included the following:

DTT (Dithiothreitol)

A 100 mM (15.4 mg/ml) solution (for use as an anti-oxidant) was made up,divided into 500 μl aliquots and stored at -20° C.

    ______________________________________                                        Hypotonic buffer:                                                             ______________________________________                                        9 mM Hepes          (214 mg/100 ml)                                           4.5% Dextran        (4.5 g/100 ml)                                            4.5 mM MgCl.sub.2   (92 mg/100 ml)                                            ______________________________________                                    

The above ingredients were dissolved in about 80 ml distilled water, pHwas adjusted to 7.8 (NaOH/HCl), the solution was then made up to 100 mlwith distilled water, and stored in a refrigerator. DTT was added to 5mM just before use (50μl/ml).

    ______________________________________                                        Isotonic buffer:                                                              ______________________________________                                        40 mM Hepes         (1.9 g/200 ml)                                            130 mM KCl          (1.94 g/200 ml)                                           4% Dextran          (8 g/200 ml)                                              2 mM EGTA           (152 mg/200 ml)                                           2.3 mM MgCl.sub.2   (94 mg/200 ml)                                            225 mM Sucrose      (15.39 g/200 ml)                                          ______________________________________                                    

The above ingredients were dissolved in about 150 ml distilled water, pHwas adjusted to 7.8 (NaOH/HCl), the solution was then made up to 200 mlwith distilled water and stored in a refrigerator. DTT was added to 2.5mM just before use (25 μl/ml).

NAD

NAD was stored as a solid in pre-weighed aliquots at -20° C. From these,solutions of a concentration of approximately 6 mM (4-4.5 mg/ml) werefreshly made up shortly before performing an assay, and the molarity waschecked by measuring the optical density (O.D.) at 260 nm. The stocksolution was then diluted with water to give a concentration of 600 μMand a small amount of ³² p labelled NAD was added (e.g. 2-5 μl/ml).

Oligonucleotide

The oligonucleotide having the palindromic sequence CGGAATTCCG,synthesised by conventional means, was vacuum dried and stored aspellets in a freezer. Before use, it was made up to 200 μg/ml in 10 mMTris/HCl, pH 7.8, with each pellet being dissolved completely in 50 mlof buffer. The solution was then heated to 60° C. in a water bath for 15minutes, and allowed to cool slowly to ensure correct reannealing. Afteradding 9.5 ml of buffer, the concentration was checked by measuring theoptical density of a diluted sample at 260 nm. The main solution wasthen diluted to a concentration of 200 μg/ml and stored in 500 μlaliquots in a freezer, ready for use.

TCA

Solutions of TCA (Trichloroacetic acid) were prepared at twoconcentrations. 10% TCA+10% sodium pyrophosphate, and 1% TCA+1% sodiumpyrophosphate.

Cells

The L1210 cells used as the source of the PARP enzyme were maintained asa suspension culture in RPMI medium+10% foetal bovine serum+glutamineand antibiotics (penicillin and streptomycin). HEPES and sodiumbicarbonate were also added, and the cells were seeded in 100 ml-200 mlof medium such that there would be a concentration of approximately8×10⁵ /ml at the time of carrying out an assay.

METHOD

The compounds being tested were generally made up as a concentratedsolution in DMSO (Dimethyl sulphoxide). The solubility of the compoundwas then checked by adding a quantity of the DMSO solution to a quantityof the isotonic buffer, in the required final proportions that were tobe used in carrying out the assay, and after an interval the solutionwas examined under a microscope for any signs of crystals forming.

A desired quantity of the cells, ascertained by counting with ahaemocytometer, was then centrifuged (1500 rpm in a "Europa" model 24Mcentrifuge for 5 minutes), the supernatant removed, and the pelletsobtained were resuspended in 20 ml Ca⁺⁺ Mg⁺⁺ free phosphate bufferedsaline (Dulbeco's modification A, abbreviated Dul A) at 4° C. beforecentrifuging again at 1500 rpm and 4° C. After again removing thesupernatant, the cells were resuspended at a concentration of 3×10⁷cells/ml in ice cold hypotonic buffer and left for 30 minutes on ice.Nine volumes were then added of ice cold isotonic buffer, and the cells,now rendered permeable to exogenous NAD⁺, were then used within the nexthour for carrying out an assay. The permeablisation of the cells may bechecked at this stage by adding duplicate aliquots of cells to an equalvolume of trypan blue, leaving for 5 minutes and then counting on ahaemocytometer. Those rendered permeable will take up the Trypan blueand appear coloured.

The assay was then carried out using for convenience plastic 15 mlconical bottomed assay tubes set up in a shaking water bath at 26° C.which is the optimum temperature for this enzyme. In a typical assayusing the oligonucleotide solution at a concentration of 5 μg/ml and thetest compound/DMSO solution at a concentration of 2%, and carrying outthe assay in quadruplicate, there would then be placed in each assaytube 5 μl of the oligonucleotide solution, 50 μl of the 600 μm NAD+[³²P]-NAD solution, 8 μl of the test compound/DMSO solution, and 37 μl ofwater. Prior to the start of the experiment this "cocktail" would bepre-warmed for 7 minutes at 26° C., as would be also the cellsuspension. The reaction would then be started by adding 300 μl of thecell suspension. The reaction would be stopped by adding 2 ml of theice-cold 10% TCA+10% sodium pyrophosphate solution.

In addition to the above, six assay tubes would usually be set up asblanks, these containing the same ingredients as above but, beforeadding the cell suspension, TCA solution is added to prevent anyreaction from taking place. This enables corrections to be applied forany non-specific binding of the labelled material to the filter used(see below).

After adding the cell suspension at timed intervals to each of the assaytubes, the 10% TCA+10% sodium pyrophosphate at 40° C. was added to eachassay tube exactly 5 minutes after addition of the cell suspension tothat tube. Then, after leaving the tubes on ice for a minimum time ofone hour, the contents of each individual tube were filtered through anindividual filter funnel of a suction filter apparatus using GF/C filterelements (rough side up) wetted with 10% TCA. After filtering thecontents of each tube and rinsing the filters several times with 1%TCA+1% sodium pyrophosphate solution, the filters were carefully removedand dried before being placed in individual scintillation vials. Fouradditional scintillation vials were also set up as reference standardscontaining 10 μl of the 600 μM NAD+[³² P]-NAD solution, 10 mlscintillant then being added to each vial. Counting was carried out for2 minutes on a β counter to obtain measures of the ³² p present, andthus the amount of the poly(ADP-ribose) and activity of the PARP enzyme.

RESULTS OF IN VITRO PARP INHIBITION STUDIES

Apart from applying the PARP enzyme assay in accordance with thestandard procedure outlined above to a range of compounds which havebeen made in accordance with the present invention, for comparisonpurposes it was also applied to certain benzamide compounds, inparticular benzamide, 3-hydroxybenzamide and 3-methoxybenzamide, thatare already known to exhibit certain PARP inhibitory activity. Atabulated list of some exemplary compounds which have been made and/orstudied is hereinafter presented in the TABLE at the end of the presentdescription, together with the PARP inhibition assay results obtained inone or more different experiments, expressed either as the percentageinhibition at a 10 μ4M concentration or, more usually, as IC₅₀ values,for the compounds when tested using the assay hereinabove described.

In reviewing this list, the known PARP inhibitors benzamide,3-aminobenzamide and 3-methoxybenzamide, may be regarded as referencecompounds. Although the results varied somewhat, in general thecompounds of the present invention which were tested showed a relativelyhigh degree of inhibitory activity. Of especial interest were thebenzimidazole carboxamides having the reference numbers NU1064, NU1066,NU1086 and, most particularly, NU1070, NU1076, NU1077, NU1085, NU1090,NU1091, NU1092, NU1093 and NU1098, of which NU1091 and NU1092 showedexceptionally high inhibitory activities.

FURTHER BIOLOGICAL ACTIVITY STUDIES

Again using cultures of the murine leukaemia L1210 cell line, growthinhibition experiments were carried out to assess the cytostatic effectsof the compounds and clonogenic survival assays were performed to assesscytotoxicity, especially in relation to use of the compounds inconjunction with DNA damaging cytotoxic agents such as cytotoxicantitumour drugs or gamma irradiation. DNA damage and the effect of thePARP inhibitors on the process of DNA strand break formation and repairhas also been assessed by carrying out DNA strand break assays andmonitoring by alkaline elution in accordance with published techniques.

In the growth inhibition assays, typically the L1210 cells would beseeded at 1×10⁴ /ml in triplicate in 24 well multidishes, and 24 hourslater the compounds or drugs being tested would be added in selectedcombinations and concentrations. At this time one set of replicateswould be counted using a Coulter counter (N₀), and 48 hours later theremaining samples would be counted (N₁). The percentage (%) growthinhibition of drug-treated samples could then be estimated. In drugcombination experiments, where evidence of synergistic effects on cellgrowth or clonogenicity was being sought, a single, fixed concentrationof a cytotoxic drug sample, e.g. temozolomide (TM), would be taken asthe control value.

Examples of in vitro Cytotoxicity Assays

In a particular example of an in vitro cytotoxicity assay using thecompound NU1064 (2-methylbenzimidazole-4-carboxamide), L1210 murineleukaemia cells were incubated with increasing concentrations of NU1064in the presence or absence of 100 μM of the methylating agent,temozolomide, in a final DMSO concentration of 1% DMSO, for 24 hours at36° C. The cells were pelleted, resuspended in fresh medium, counted andseeded for colony formation in 0.15% agarose in drug-free medium. After1 week colonies of viable cells were stained with MTT (1 ml 0.5 mg/ml)and counted. The plating efficiency of the control (89%) andtemozolomide alone (32%) were normalised to 100% relative survival andthe plating efficiency of the NU1064-treated cells expressed as apercentage of these values.

There was a modest reduction in cell survival caused by NU1064 alone(relative plating efficiency at 100 μM and 200 μM NU1064=72% and 54%,respectively) but a very marked increase in temozolomide cytotoxicitywith increasing concentrations of NU1064 (relative plating efficiency at100 and 200 μM NU1064=28% and 2%, respectively) indicating aNU1064-concentration-related potentiation of temozolomide cytotoxicity.An illustration of these results is presented by FIG. 1 of theaccompanying drawing.

In other, clonogenic survival, assays, typically the L1210 cells wouldbe exposed to varying concentrations of TM±a fixed concentration of PARPinhibitor for a fixed time of 16 hours, prior to counting and seedingfor colony formation in 0.12-0.15% agarose in drug-free medium. After7-10 days colonies would be stained with 0.5 mg/ml MTT and counted byeye on a gridded light box. This then enables survival curves to beplotted and DEF₁₀ values to be obtained, DEF₁₀ being defined as theratio of the concentration of TM that reduces survival to 10% divided bythe concentration of TM that reduces survival to 10% in the presence ofa fixed concentration of PARP inhibitor.

In further clonogenic survival assays gamma ray irradiation may be usedto damage the cells. Typically, L1210 cells (3 ml, 4×10³ /ml in plasticbijoux bottles) would be irradiated at 4° C. with varying doses of gammarays in the presence or absence of the compound being tested and a finalconcentration of 2% DMSO. The cells would then be incubated at 37° C.for 2 hours in the continued presence or absence of PARP inhibitor priorto seeding for colony formation.

Repair of potentially lethal damage (PLD) occurs when cells are held instationary-phase following initiation of PLD prior to allowing celldivision to take place. In further typical experiments to test potentialPARP inhibitors, L1210 cells have been allowed to repair gamma ray PLDin the presence or absence of the test compound as follows: L1210 cellswere maintained in culture until they had attained stationary phase(>10⁶ cells/ml). They were diluted to 1.5×10⁵ /ml in conditioned mediumfrom stationary-phase cultures to prevent further cell division.Replicate 2 ml samples of cells in plastic bijoux were held on ice priorto and immediately following 8 Gray gamma ray irradiation. 1 ml of 3×final concentration of the test compounds made up in conditioned mediumfrom stationary cultures would then be added to give appropriate finalconcentrations (e.g. 10⁶ cells/ml in 1% DMSO±test compounds), and thecells would be incubated at 37° C. for 0, 2 or 4 hours prior toresuspending in drug-free medium and seeding for colony formation.Unirradiated stationary phase cultures incubated at 37° C. for 0, 2 or 4hours with 1% DMSO±the same amount of test compound provide appropriatecontrols for determining relative cell survival. In the absence of PARPinhibitor cell survival would normally increase with time allowed forPLD repair to take place. For example, in one set of experiments, whenseeded immediately after irradiation (no repair) only about 0.2% of thecells survived, but after a 4 hour repair period this had increased to0.7%. An effective PARP inhibitor blocks this repair, thus reducing thesurvival rate.

With regard to the DNA strand break assays previously mentioned,typically samples of L1210 cells would be incubated for a certain time,e.g. 1 hour, with a fixed concentration, e.g. 150 μM, of temozolomideand, apart from a control, in the presence of increasing concentrationsof the PARP inhibitors tested. The more effective the inhibitor, thegreater the rate of the alkaline elution (a measure of extent of strandbreakage) compared to temozolomide alone.

In general, the studies carried out fully support the belief that thePARP inhibitory characteristics of the compounds tested reflect anability of these compounds to potentiate the cytotoxicity of DNAdamaging agents, such as certain cytotoxic antitumour drugs andradiation used in radiotherapy. Accordingly, having regard to theirstrong PARP inhibitory characteristics, the compounds of this inventioncan be expected to be especially useful for administration inconjunction with such cytotoxic drugs or radiotherapy in order topotentiate the cytotoxic effect of the latter in the course of medicaltreatment as hereinbefore indicated.

SUMMARY

Although the present invention should be regarded overall as comprisingeach and every novel feature or combination of features disclosedherein, the main aspects of the invention comprise, principally but notexclusively, broadly the following:

(i) Novel compounds of formula (I) as defined herein;

(ii) Compounds of formula (I) with substituents as hereinbefore defined(including pro-drug forms and salts thereof) for therapy or for use inmedicine and in the manufacture of medical preparations, useful forexample as PARP inhibitors to be administered in conjunction withcytotoxic drugs or with radiotherapy to potentiate the effectiveness ofthe latter in treatment of cancer;

(iii) Processes for the preparation of novel compounds of formula (I) asdefined herein, including any novel intermediate compounds produced incarrying out such processes;

(iv) Pharmaceutical formulations comprising a compound of formula (I) asdefined herein together with a pharmaceutically acceptable carriertherein; and

(v) Processes for the preparation of a pharmaceutical formulation asdefined in (iv) above, e.g. by methods referred to herein.

    __________________________________________________________________________                                         % Inhibition at                          House No.                                                                          Name        Structure           10 μM or IC.sub.50                    __________________________________________________________________________                                         value                                    Ref  benzamide  C.sub.7 H.sub.7 O  MW = 121.1                                                   ##STR6##           IC.sub.50 = 12.4 ± 3.1 μM          Ref  3-hydroxybenzamide  C.sub.7 H.sub.7 NO.sub.2  MW = 137                                     ##STR7##           IC.sub.50 = 8.0 ± 3.5 μM (7)       Ref  3-methoxybenzamide  C.sub.8 H.sub.9 NO.sub.2  MW = 151                                     ##STR8##           55                                       NU1064                                                                             2-methylbenzimidazole-4-  carboxamide  C.sub.9 H.sub.9 N.sub.3 O  MW          = 175.38                                                                                   ##STR9##           IC.sub.50 = 1.09 ± 0.23 μM                                              (3)                                      NU1066                                                                             benzimidazole-4-  carboxamide  C.sub.8 H.sub.7 N.sub.3 O  MW =                161.16                                                                                     ##STR10##          IC.sub.50 = 1.26 μM  IC.sub.50 =                                           1.02 μM                               NU1067                                                                             benzimidazole-4-  carboxylic acid  C.sub.8 H.sub.6 N.sub.2 O.sub.2            162.14                                                                                     ##STR11##          Inactive                                 NU1070                                                                             2-phenylbenzimidazole-4-  carboxamide  C.sub.14 H.sub.11 N.sub.3 O            237.26                                                                                     ##STR12##          IC.sub.50 = 92 nM  IC.sub.50 = 103                                            nM                                       NU1076                                                                             2-(4-methoxyphenyl)  benzimidazole-4-  carboxamide  C.sub.15                  H.sub.13 N.sub.3 O.sub.2  267.28                                                           ##STR13##          IC.sub.50 = 59 nM                        NU1077                                                                             2-(4-trifluoro-  methylphenyl)  benzimidazole-4-  carboxamide                 C.sub.15 H.sub.10 N.sub.3 OF.sub.3  305.25                                                 ##STR14##          IC.sub.50 = 75 nM                        NU1085                                                                             2-(4-hydroxyphenyl)  benzimidazole-4-  carboxamide  C.sub.14                  H.sub.11 N.sub.3 O.sub.2  253.26                                                           ##STR15##          IC.sub.50 = 77 nM                        NU1086                                                                             2-trifluoromethyl-  benzimidazole-4-  carboxamide  C.sub.16 H.sub.15          N.sub.3 O.sub.2  281.31                                                                    ##STR16##          IC.sub.50 = 1.6 μM                    NU1090                                                                             2-(4-methoxyphenyl)-N-  methylbenzimidazole-4-  carboxamide                   C.sub.16 H.sub.15 N.sub.3 O.sub.2  281.31                                                  ##STR17##          IC.sub.50 = ˜100 nM                NU1091                                                                             2-(4-nitrophenyl)-  benzimidazole-4-  carboxamide  C.sub.14 H.sub.10          N.sub.4 O.sub.3  282.25                                                                    ##STR18##          IC.sub.50 = 22 nM                        NU1092                                                                             2-(4-cyanophenyl)-  benzimidazole-4-  carboxamide  C.sub.14 H.sub.10          N.sub.4 O  262.27                                                                          ##STR19##          IC.sub.50 = 33 nM                        NU1093                                                                             2-(3-trifluoromethyl-  phenyl)benzimidazole-4-  carboxamide                   C.sub.15 H.sub.10 N.sub.3 OF.sub.3  305.25                                                 ##STR20##          IC.sub.50 = 76 nM                        NU1098                                                                             2-(3-methoxyphenyl)  benzimidazole-4-  carboxamide  C.sub.15                  H.sub.13 N.sub.3 O.sub.2  267.28                                                           ##STR21##          IC.sub.50 = 130 nM                       NU1101                                                                             N-benzoyl-2-(4-  methoxyphenyl)-  benzimidazole-4-  carboxamide               C.sub.22 H.sub.17 N.sub.3 O.sub.3  371.39                                                  ##STR22##          IC.sub.50 = 0.27 μM                   NU1103                                                                             2-(4-aminophenyl)-  benzimidazole-4-  carboxamide  C.sub.14 H.sub.12          N.sub.4 O  252.27                                                                          ##STR23##          IC.sub.50 = 91 nM                        NU1104                                                                             2-(2-trifluoromethyl-  phenyl)benzimidazole-4-  carboxamide                   C.sub.15 H.sub.10 N.sub.3 OF.sub.3  305.25                                                 ##STR24##          Not tested                               NU1105                                                                             N-carboxybenzyl-2-(4-  methoxyphenyl)-  benzimidazole-4-                      carboxamide  C.sub.23 H.sub.19 N.sub.3 O.sub.4  401.42                                     ##STR25##          1.9 μM                                __________________________________________________________________________

What is claimed is:
 1. A pro-drug of a compound having the structuralformula I ##STR26## wherein R is selected from the group consisting ofhydrogen, hydroxyalkyl and benzyl and phenyl groups which areunsubstituted or substituted by at least one substituent in the benzenering selected from the group consisting of hydroxy, alkoxy, NO₂, N₃, NR₅R₆ (R₅ and R₆ each being independently hydrogen, alkyl or alkoxy),NHCOR₃ (R₃ being alkyl or aryl), CO₂ R₄ (R₄ being H or alkyl), an amide,tetrazole, alkyl, hydroxyalkyl, CW₃ or W (W being halogen), and CN,andR' is selected from the group consisting of hydrogen, alkyl,hydroxyalkyl, acyl or an optionally substituted phenyl group having thestructural formula III: ##STR27## wherein R₇, R₈ and R₁₀ are eachselected independently from the group consisting of H, hydroxy, alkoxy,NO₂, NO₃, NR₅ R₆ (R₅ and R₆ each being independently hydrogen, alkyl oralkoxy), NHCOR₃ (R₃ being alkyl or aryl), CO₂ R₄ (R₄ being H or alkyl),an amide, tetrazole, alkyl, hydroxyalkyl, CW₃ or W (W being halogen),and CNwherein the pro-drug form is a phosphate derivative of saidcompound.
 2. A compound as claimed in claim 1 said compound being in theform of a phosphate pro-drug provided by a water-soluble ammonium oralkali metal phosphate salt of said benzimidazole compound of structuralformula I having at least one hydroxyl group substituent.
 3. Apharmaceutical composition comprising an effectivepoly(ADP-ribose)polymerase or PARP enzyme inhibiting amount of abenzimidazole compound having the structural formula I ##STR28## or apharmaceutically acceptable salt and/or pro-drug form thereof, togetherwith a pharmaceutically acceptable carrier,wherein R is selected fromthe group consisting of methyl, ethyl, n-propyl, i-propyl, n-butyl,t-butyl and cyclohexyl, and R' is selected from the group consisting ofhydrogen, alkyl, hydroxyalkyl, acyl and an optionally substituted phenylgroup having the structural formula III: ##STR29## wherein R₇, R₈ andR₁₀ are each selected independently from the group consisting of H,hydroxy, alkoxy, NO₂, N₃, NR₅ R₆ (R₅ and R₆ each being independentlyhydrogen, alkyl or alkoxy), NHCOR₃ (R₃ being alkyl or aryl), CO₂ R₄ (R₄being H or alkyl), an amide, tetrazole, alkyl, hydroxyalkyl, CW₃ or W (Wbeing halogen), and CN.
 4. A pharmaceutical composition comprising aneffective poly (ADP-ribose) polymerase or PARP enzyme inhibiting amountof a benzimidazole compound having the structural formula I ##STR30## Inpro-drug form, together with a pharmaceutically acceptable carrier,whereinR is selected from the group consisting of hydrogen, alkyl,hydroxyalkyl and benzyl and phenyl groups which are unsubstituted orsubstituted with at least one substituent in the benzene ring selectedfrom the group consisting of hydroxy, alkoxy NO₂, N₃, NR₅ R₆ (R₅ and R₆each being independently hydrogen, alkyl or alkoxy), NHCOR₃ (R₃ beingalkyl or aryl), CO₂ R₄ (R₄ being H or alkyl), an amide, tetrazole,alkyl, hydroxyalkyl, CW₃ or W (W being halogen), and CN, and R' isselected from the group consisting of hydrogen, alkyl, hydroxyalkyl,acyl and an optionally substituted phenyl group having the structuralformula III: ##STR31## wherein R₇, R₈ and R₁₀ are each selectedindependently from the group consisting of H, hydroxy, alkoxy, NO₂, N₃,NR₅ R₆ (R₅ and R₆ each being independently hydrogen, alkyl or alkoxy),NHCOR₃ (R₃ being alkyl or aryl), CO₂ R₄ (R₄ being H or alkyl), an amide,tetrazole, alkyl, hydroxyalkyl, CW₃ or W (W being halogen), andCNwherein the pro-drug is a phosphate derivative of a compound havingthe structural formula I.
 5. A pharmaceutical composition as claimed inclaim 4 wherein said pro-drug form is provided by a water-solubleammonium or alkali metal phosphate salt derived from a compound ofstructural formula I that has at least one hydroxyl group substituent.6. A pharmaceutical composition as claimed in claim 5 wherein thecompound of structural formula I from which the phosphate pro-drug isderived has a hydroxyl group substituent that reacts with a dibenzylphosphonate.
 7. A method of improving the effectiveness of a cytotoxicdrug or radiotherapy in the course of antitumor therapy on a mammalwhich comprises administering to said mammal, in conjunction with theadministration of said drug or radiotherapy, an effectivePARP-inhibiting amount of a compound having the structural formula I##STR32## or a pharmaceutically acceptable salt or pro-drug thereof,whereinR is selected from the group consisting of hydrogen, hydroxyalkyland benzyl and phenyl groups which are unsubstituted or substituted byat least one substituent in the benzene ring selected from the groupconsisting of hydroxy, alkoxy, NO₂, NO₃, NR₅ R₆ (R₅ and R₆ each beingindependently hydrogen, alkyl or alkoxy), NHCOR₃ (R₃ being alkyl oraryl), CO₂ R₄ (R₄ being H or alkyl), an amide, tetrazole, alkyl,hydroxyalkyl, CW₃ or W (W being halogen), and CN, and R' is selectedfrom the group consisting of hydrogen, alkyl, hydroxyalkyl, acyl or anoptionally substituted phenyl group having the structural formula III:##STR33## wherein R₇, R₈ and R₁₀ are each selected independently fromthe group consisting of H, hydroxy, alkoxy, NO₂, N₃, NR₅ R₆ (R₅ and R₆each being independently hydrogen, alkyl or alkoxy), NHCOR₃ (R₃ beingalkyl or aryl), CO₂ R₄ (R4 being H or alkyl), an amide, tetrazole,alkyl, hydroxyalkyl, CW₃ or W (W being halogen), and CN.
 8. A method ofimproving the effectiveness of a cytotoxic drug or radiotherapy in thecourse of antitumor therapy on a mammal which comprises administering tosaid mammal, in conjunction with the administration of said drug orradiotherapy, a PARP enzyme inhibiting amount of a benzimidazolecompound having the structural formula I ##STR34## or a pharmaceuticallyacceptable salt and/or pro-drug form thereof, whereinR is selected fromthe group consisting of hydrogen, alkyl, hydroxyalkyl, acyl and anoptionally substituted aryl or aralkyl group, and R' is selected fromthe group consisting of hydrogen, alkyl, hydroxyalkyl, acyl and anoptionally substituted aryl or aralkyl group.
 9. A method of improvingthe effectiveness of a cytotoxic drug or radiotherapy in the course ofantitumor therapy on a mammal which comprises administering to saidmammal, in conjunction with the administration of said drug orradiotherapy, a PARP enzyme inhibiting amount of a benzimidazolecompound selected from the group consistingof2-methylbenzimidazole-4-carboxamide; benzimidazole-4-carboxamide;2-(4'-methoxyphenyl)benzimidazole-4-carboxamide;2-(4'-trifluoromethylphenyl)benzimidazole-4-carboxamide;2-(4'-hydroxyphenyl)benzimidazole-4-carboxamide;2-trifluoromethylbenzimidazole-4-carboxamide;2-(4'-methoxyphenyl)-N-methylbenzimidazole-4-carboxamide;2-(4'-nitrophenyl)benzimidazole-4-carboxamide;2-(4'-cyanophenyl)benzimidazole-4-carboxamide;2-(3'-trifluoromethylphenyl)benzimidazole-4-carboxamide;2-(3'-methoxyphenyl)benzimidazole-4-carboxamide;2-(4'-methoxyphenyl)-1-N-benzoylbenzimidazole-4-carboxamide;2-(4'-aminophenyl)benzimidazole-4-carboxamide;2-(2'-trifluoromethylphenyl)benzimidazole-4-carboxamide; andN-carboxybenzyl-2-(4'-methoxyphenyl)-benzimidazole-4-carboxamide.